Requirements of Cellular Survival and Proliferation

Mark A. Carlson

UNMC Dept Surgery Grand Rounds, September 3, 2003

Overview:

• proliferation (cell cycle)• cell death • basic requirements• cell anchorage• growth factor dependence• cell shape• granulation tissue survival and proliferation• fibroblast survival in a 3-D matrix

Requirements of Cellular Survival and Proliferation

Approximate number of cells in the human body:50 trillion (50,000,000,000,000)

Cells undergoing apoptosis per day (and replaced):10 billion (< 0.1%)

Interesting facts & figures

Interesting facts & figures (cont’d)

“With an estimated mutation rate of some 1 in 2 × 107

per gene cell division, some 1014 target cells in the averagehuman, and an abundant repertoire of genes regulating allaspects of cell expansion, it is remarkable that cancers arisein only 1 in 3 lifetimes.”

(Evan & Vousden, Nature 2001;411:342)

Proliferation control: the cell cycle

(Milde-Langosch and Riethdorf, J Cell Physiol 2003;196:224)

Cell cycle:G1 checkpoint

(Stewart et al., Trends Pharmacol Sci 2003;24:139)

Cell cycle: G2 checkpoint

(Stewart et al., Trends Pharmacol Sci 2003;24:139)

(University of Arizona Biology Project web site)

Mitosis

[go to “Vid1_mitosis.mov”]

Mitosis

(University of North Carolina Chapel Hill Department of Biology web site)

[go to “Vid2_BloodLilyMitosis.mov”]

Types of cell death

• necrosis

• apoptosis(anoikis)

• nonapoptotic, nonnecrotic cell death (?)

• fixation

Necrosis (“cell murder”)

• sudden disruption of homeostasis

• swelling, cell lysis

• inflammatory infiltrate

Acute myocardial infarction

(University of Illinois College of Medicine Urbana-Champaign web site)

Necrosis (viral hepatitis)

(Curran, Color Atlas of Histopathology, 1985)

Apoptosis (“cell suicide”)

• cell shrinkage

• blebbing/phagocytosis

• no inflammation

Trophoblast apoptosis

(University of London St. George’s Hospital web site)

[Go to “Vid3_TrophoblastApoptosis.mov”]

Apoptosisvs.

necrosis

(Karolinska Institute web site)

HeLa cell apoptosis

( Morgan et al., J Cell Biol 2002;157:575)

[Go to “Vid4_HeLaApoptosis.mov”

ApoptoticSignalingPathways

p53 activators

Multiplicity of p53 effects

(Manfredi, Mol Cell 2003;11:552)

(Brooks & Gu, Curr Opin Cell Biol 2003;15:164)

Regulation of p53

ApoptoticSignalingPathways

protein kinase B (PKB, or Akt)

central role in survival signaling& cell cycle regulation

(Brazil et al., Cell 2002;111:293)

Akt interactions

Cell survival & proliferation:Basic requirements

• hydration• ionic• O2• pH• temperature• substrate

• growth factors• adhesion (anchorage dependence)• shape

Cell survival & proliferation:Cell-specific requirements

Human umbilical veinendothelial cells grownon a culture dish

Fujio & Walsh, J Biol Chem 1999;274:16349

Growth factor signaling(receptor tyrosine kinase)

(Garrett & Grisham, Biochemistry, 2nd Ed.)

Anchorage-dependent survival & proliferation

Anoikis (detachment-induced apoptosis)

Renal epithelial (MDCK) cellsgrown in suspension (noanchorage to a substratum)

Khwaja et al., EMBO J 1997:16:2783

Most primary cells require anchorage for survival

Exceptions:

• leukocytes• lymphocytes• monocytes

(i.e., hematogenous cells)

• gamete cells (spermazoa, ova)

Chen et al., Science 1997;276:1425

Dependence of cell growth& survival on cell shape

Human pulmonary capillary endothelial cellsgrown on fibronectin-coated beads

Shape

Chen et al., Science 1997;276:1425

Cell shape (cont’d)

Malignant cells

• Anchorage independence(e.g., growth in soft agar)

• Metastatic potential• GF independence• Shape?

good

tissue loss

response

DEATH

healing

regenerationscar withdisability

scar with minimal or no disability

(quality of life scale)poor

Figure 1: overview of response to tissue loss. A mammal will respond to nonfatal tissue loss with healing or with (in select tissues) regeneration. Healing results in scar formation which, depending on the size and location of the tissue loss, can produce disability, poor quality of life, and premature death.

– – – –

Wound Healing ResearchResponse to tissue loss

Burn wound contracture

(Longacre, 1972)

Excisional wound model

5 min after excision postwounding day 5

GT

Interesting facts & figures in healing

Number of wound cells immediately post-excision: zero

Number of wound cells on PWD 5: ~200 million

Number of cells in scar (PWD 30): < 1 million

My research:

A focus on the intermediate organof healing (= granulation tissue)

Granulation tissue

(open abdomen)

Regulation of granulation tissueproliferation and survival

Wound irrigationduring granulationtissue formation

Neutralization of PDGF duringgranulation tissue formation

Wound splinting model

Effect of wound splinting on granulation tissue proliferation

effect of wound anchorage on granulation tissue survival

Wound flap model

Chronic inhibition by a flap

Moderation of flap effect withimplanted impermeable barrier

Flap-induced granulationtissue regression

Fibroblast survival & proliferation in the collagen matrix

Three-dimensional vs.two-dimensional culture systems

• 2-D: monolayer• 3-D: fibroblast-populated collagen

matrix (FPCM)• in vivo systems (animal models)

(Cukierman et al., Science 2001;294:1708)

2D vs. 3D adhesions

The anchored fibroblast-populated collagen

matrix: a model of granulation tissue

attached/mechanically ! loaded (isometric tension) ! ! granulation tissue

released/mechanically ! unloaded

! scar

1 day

detach with spatula

1 day

dissipation of mechanical ! load

Wound model: fibroblast-populated

collagen matrix

(Comparison of thewound and FPCM)

Is the FPCM a good model?

Comparison of models (cont’d)

Fibroblast-populated collagen matrix

Nakagawa et al., J Invest Dermatol 1989;93:792

0 10 20 30 40 500

5

10

15

20

released

attached

incubation time (hr)

%apoptotic

Apoptosis in attached vs. released collagen matrices

*

*

(Grinnell et al., Exp Cell Res 1999;248:648)

ECM

!"

FAK

GF

GFR

PI3K

PI3K

Apoptotic pathways

nucleus

Attached

Akt

p53

FAK

GF

GFR

death

Akt

p53

!"

ECM

Apoptotic pathways

Detached

Regulation of fibroblast survival in the collagen matrix

FAK and Akt activity in the FPCM

ECM

!"

FAK

GF

GFR

PI3K

PI3K

Apoptotic pathways

nucleus

Attached

Akt

p53

FAK

GF

GFR

death

Akt

p53

!"

ECM

Apoptotic pathways

Detached

Regulation of fibroblast survival in the collagen matrix

0 3 60

7500

15000

0

1

2

0 3 6

FAKtub

FAK/tubD

unitsFAK/tub

ratio

FAK

tubulin

post transfection day

vehicle only vehicle + siRNA

Figure A-1: RNA interference of FAK in foreskin fibroblasts. Cells (24-well plates; 10,000 cells per well) were treated with 200 nM of FAK-specific siRNA duplex in 0.5% lipid vehicle for 4 hr. Lysates were made on days 1-6 post-transfection, and immunoblotted for total FAK and α-tubulin. Relative FAK expression was calculated from densiometry (D) of the blots (FAK:tubulin band ratio).

RNA interference (RNAi)

Figure A2: RNAi of FAK in the FPCM induces cell death. Matrices were incubated in the attached state for 72 hr prior to transfection with 67 nM of FAK-specific siRNA duplex in 0.67% of lipid transfection vehicle for 4 hr. The cell were retrieved after enzymatic degradation of the matrix, and TUNEL with PI counterstain was performed on cytospin preparations. Each bar represents the average of 4 fields. The experiment was performed twice with similar results.

day post-transfection

%apop

2 3 60

5

10

15

20–siRNA+siRNA

RNAi of FAKin the FPCM

ECM

!"

FAK

GF

GFR

PI3K

PI3K

Apoptotic pathways

nucleus

Attached

Akt

p53

FAK

GF

GFR

death

Akt

p53

!"

ECM

Apoptotic pathways

Detached

Regulation of fibroblast survival in the collagen matrix

p53, mdm2, p21 activityin monolayer & the FPCM

monolayer

FPCM

p53 FITC PI

att

rel

p53 FITC PI

transfection: vehicle only vehicle + siRNA

RNAi of p53 in attached vs. released FPCM(p53 immunohistochemistry)

ECM

!"

FAK

GF

GFR

PI3K

PI3K

Apoptotic pathways

nucleus

Attached

Akt

p53

FAK

GF

GFR

death

Akt

p53

!"

ECM

Apoptotic pathways

Detached

Regulation of fibroblast survival in the collagen matrix

p53 IHC (% positive) TUNEL (%positive)

transfection attached detached attached detached

non 3.2 ± 1.9 27.3 ± 4.4* 0.5 ± 0.7 10.7 ± 3.6*

vehicle only 7.9 ± 5.0 26.0 ± 7.5* 1.7 ± 1.3 12.4 ± 2.1*

RNA duplex 8.4 ± 2.0** 15.0 ± 1.5*,** 5.0 ± 2.6** 4.7 ± 1.9**

TUNEL PI

att

rel

TUNEL PI

transfection: vehicle only vehicle + siRNA

RNAi of p53 in attached vs. released FPCM(TUNEL assay)